ABSTRACT UTX is a chromatin modifier required for the development of brain, heart, and bone. To facilitate gene activation, UTX removes methylation from methylated lysine 27 in histone H3 (H3K27 methylation) and promotes H3K27 acetylation, H3K4 methylation, and open chromatin structure. In humans, UTX mutations are causally linked to a developmental syndrome and to many childhood and adult cancers of the brain, blood, bladder, esophagus, kidney, and breast. Although the importance of UTX is established, how it targets and regulates genes remains unclear. In particular, contradictory findings raise the question about which chromatin modifying activity of UTX is important for developmental gene regulation in stem cells. This knowledge gap limits our understanding of the etiology of developmental defects and cancers associated with UTX dysfunction or H3K27 modifications. Our long-term goal is to fill this knowledge gap by determining how UTX regulates chromatin structure and gene expression to govern stem cell functions. Our preliminary studies identified a protein network of UTX that is important for the differentiation of human pluripotent stem cells to the neural lineage. In this network, DNA damage response factors play a noncanonical role in regulating gene expression. Our central hypothesis is that this UTX-centric network facilitates chromatin changes and transcriptional activation during stem cell differentiation. To test this hypothesis, we plan to identify the chromatin-regulatory activity of UTX that affects transcription, examine the noncanonical function of DNA damage response factors in this network, and elucidate the role of a downstream effector that executes gene expression programming. Our approaches will take advantage of the conceptual innovation about a new UTX-driven protein network and the technological innovation of combining Cas9-CRISPR for structure?function studies, genomics assays, and the human cortical organoid model. If successful, we expect our findings to have wide implications on epigenetic regulation of human stem cells in development and cancer.